Lymphocyte Antigen Receptor Diversity

Lymphocyte Antigen Receptor Diversity

• B cell and T cell receptors, or antibodies, are designed to recognize a wide array of antigens.
• Somatic recombination, a process where small DNA regions are cut and pasted back together, creates diverse receptors with antigen specificities.
• Antibodies have 2 heavy and 2 light chains, each containing a variable and at least one constant region.
• The variable heavy and light chain domains create the antigen-binding site.
• Variations in the amino acid sequence of the antigen-binding site allows for a vast range of antigen receptors.
• Variable regions utilize an Immunoglobin-fold structure that consists of 9 beta strands, creates 2 beta sheets, and is held together via a disulfide bond, featuring 3 loops, hypervariable regions (HV), or complementary determining regions (CDRs).
• CDRs come into contact with the antigen and define the antigen-binding site's specificity.
• The diversity of antibodies is generated in these regions.
• Antibodies and B/T Cell Receptors are expressed as proteins, following the central dogma of biology.
• Somatic recombination occurs during B and T cell development, happens at the DNA level, and is irreversible.
• Changes are passed to progeny.

Heavy Chain Variable Domain Rearrangement Notes

• A typical heavy chain contains 1 variable and 3 constant domains, and the gene encoding a heavy chain has 4 exons.
• The "exon" coding for the variable domain is segmented into V, D, and J parts which must be joined together via somatic recombination for a functional variable domain.
• Somatic recombination takes place in heavy chains via 2 steps: 1) D joining to J, and 2) V joining to DJ.
• After somatic recombination, the gene is transcribed to primary RNA, processed (spliced), and resultant mRNA is translated to protein in the cytosol.

Light Chain Variable Domain Rearrangement info

• Light Chain Variable Domain Rearrangements occur in the same manner as heavy chains, but there is no D segment, so it is just 1 step connecting V to J.

Combinatorial Diversity Facts

• Complete variable domain has multiple copies of each V, D, and J segment, each having individual sequences.
• Diversity of the variable domains are created by the random selection of 1 segment of each type.

Combinatorial Diversity Points

• Stems from the combination of heavy and light chains.
• There are 2 types of light chains, Kappa and Lambda.
• In each chain, the variable domain has multiple V and J segments.
• Around 1.5 million different antibodies can be produced through this combination.

VDJ Recombination Process - Mechanism

• The somatic recombination process is specific to VDJ segments.
• Cells do not recombine DNA under normal circumstances as abnormal gene products can result and lead to cancer.
• Somatic recombination has to take place at the correct place in a regulated way.
• V segments must join to D or J, and not another V.
• Recombination Signal Sequences (RSS) are conserved, non-coding DNA sequences that guide VDJ recombination.
• Segments that are to be recombined are adjacent to RSS.
• The RSS features include: Heptamer (7bp) that is close to the coding region of the segment, Nonamer (9bp) that is away from the coding region, and a Spacer in between that is either 23 or 12 bp long.
• Recombination occurs between segments in the same chromosome, following a 12/23 rule where an RSS with a 12bp recombines with an RSS with a 23bp.

VDJ recombination machinery explained

• RSS are identified via components of the recombination machinery, or Recombinases.
• Recombinases, RAG1 and RAG2, are only expressed in lymphoid cells during development. They form a heterotretamer complex of 2 RAG1 and 2 RAG2.
• The RAG1 section identifies and binds to the RSS, and it has endonuclease activity with RAG2 as its cofactor.
• The Non-homologous End joining complex (NHEJ) is involved in DNA double-strand break repair, and it utilizes an imprecise mechanism that leads to gain or loss of nucleotides.
• Ku is a heterodimer comprised of Ku70:Ku80, forms a ring around the DNA.
• DNA Protein kinase (DNA-PK) couples with the Ku complex, phosphorylates and activates Artemis
• Artemis has nuclease activity and creates palindromic sequences.
• Terminal deoxynucleotidyl Transferase (TdT) randomly adds nucleotides.
• DNA Ligase IV joins the DNA ends together.

Somatic Recombination Steps

1. RAG1/2 complex binds sequentially to 2 RSS, making a precise single strand cut between the RSS and the coding region, creates a hairpin at the coding region.
2. KU complex creates a ring around the DNA.
3. KU then recruits DNA-PK which then recruits Artemis.
4. DNA-PK phosphorylates Artemis, this is what cuts the hairpin.
5. Terminal deoxynucleotidyl Transferase (TdT) adds nucleotides to the single strands.
6. DNA ligase IV joins the ends.

Junctional Diversity Details

• HV1 (CDR1) and HV2 (CDR2) are of the 3 hypervariable loops of the immunoglobin are coded by V segments whereas HV3 (CDR3) is coded by the joint of V and D or V and J.
• During segment joining, nucleotides are added and removed, which creates a large amount of diversity in the immunoglobulin molecule, junctional diversity.

Molecular Mechanism of Junctional Diversity

• RAG1/2 activity creates DNA hairpins that are adjacent to the coding region of the VDJ segments.
• After being phosphorylated via DNA-PK, Artemis slices hairpins to generate palindromic nucleotides (P-nucleotides).
• TdT adds random nucleotides (N-nucleotides), up to 20, to the end of the single-strand segments.
• The single strands then pair, unpaired nucleotides are removed via an exonuclease.
• Gaps are filled by DNA polymerase and are joined using DNA ligase IV.

Generation of the Constant Region

• The heavy chain locus encodes multiple constant regions that are present as separate genes.
• Each constant region gene encodes for a different antibody isotype, and each constant gene is split into exons corresponding to each Ig domain.
• B cells initially express 2 antibody isotypes on their surface, IgM and IgD.
• B cells produce a long primary RNA, which is differentially spliced to produce either of the 2 mRNAs products.

Membrane-bound vs Secreted Immunoglobins

• Membrane-bound form: The heavy chain has a hydrophobic transmembrane domain that anchors it to the B cell.
• Secreted form: Replaces transmembrane domain for a hydrophilic secretory tail.
• Both the transmembrane domain and the secretory tail are encoded by different exons at the end Constant region gene, and go through alternative splicing.

T Cell Receptor Somatic Recombination

• Alpha chains = light chains, and Beta chains = heavy chains, this process is the same as immunoglobin recombination.
• Always membrane-bound.
• Always 1 constant region.
• D segments have both 12 and 23 bp spacers
• Sometimes D segments may or may not be included.

Antigen Presentation to T cells

• TCRs recognize antigens that are processed and then presented to them.
• TCRs recognize peptide antigens, which are otherwise buried within a protein that needs to be broken down into fragment peptides.
• Then, peptide antigens are loaded onto MHC molecules, and they are presented at the cell surface.
• Peptides recognized by TCR derive from the turnover of normal proteins, intracellular pathogens (viruses), or extracellular fluids.
• MHC class I binds peptides that are recognized by CD8 T Cells (Cytotoxic T cells).
• MHC class I is expressed in all nucleated cells and CD8 T cells are in charge of surveillance.
• CD8 T cells kill nucleated cells infected with intracellular pathogens (viruses or bacteria), and also recognize cancer cells.
• MHC class II binds peptides that are recognized by CD4 T cells (Helper T Cells).
• MHC class II is expressed in antigen-presenting cells (Dendritic cells, B cells, and macrophages).
• Antigens presented by MHC class II stem from extracellular fluid internalized by phagocytosis and endocytosis.
• CD4 T cells activate other cells of the immune system.

Antigen Derivation in Cells

• Main cellular compartments:
  • Cytosol, contiguous to the nucleus, is loaded to class I, mostly with viruses.
  • Vesicular system, is involved in endocytosis and secretion, loaded to class II, and has pathogens that survive inside phagosomes.
• Before being loaded onto MHC class I molecules, peptide antigens are generated intracellularly using degradation of proteins.
• Cytosolic proteins are degraded by the proteasome, which forms a multi-protein complex.
• The proteasome consists of 2 19S regulatory caps and a 20S catalytic core, the regulatory caps have a lid and a base to recognize substrates and unfold proteins.
• The catalytic core consists of alpha and beta subunits, beta subunits have protease activity and degrade proteins.
• Proteins are first labeled with ubiquitin chains by E3 Ubiquitin ligases when they are targeted for proteasomal degradation.
• During infection, in response to INF- gamma, the proteasome alters its composition. The catalytic core then yields peptides that bind the MHC class I more efficiently.

Synthesis and Assembly of MHC Class I

• Proteins (alpha and B2-microglobulin) that create Class I are translocated into the ER during synthesis, and then folded into a complex.
• Calnexin (an ER protein) retains improperly folded alpha chains for proper MHC molecule folding so that binding of the peptide antigen can occur.
• Calnexin releases the assembled MHC molecule after binding of B2-microglobulin, which is then bound by the Peptide Loading Complex (PLC).
• The complex has 2 jobs: retaining MHC in the ER and keeping it folded until the antigen peptide is loaded.
• PLC is made of 2 chaperones, Calreticulin, ERp57, and Tapasin, that bridges PLC to TAP.
• The proteasome creates peptides inside the cytosol, and TAP (a heterodimer) will translocate them into the ER.
• Some peptides that are translocated will directly bind to the MHC molecule, while others are trimmed by peptidase ERAAP.
• Once loaded with a peptide, the peptide:MHC complex is released and transported to the cell surface.
• MHC class I molecules are loaded with peptides that are derived from normal "old" proteins and Defective Ribosomal Products, such as peptides translated from introns, frameshifts, improperly folded peptides.

Processing and Presentation of Extracellular Antigens

• Antigens are taken up from extracellular space during endocytosis/phagocytosis.
• Protesases are activated via the early endosome which causes antigen degradation.
• Lysosomes fuse with the early endosome.
• Peptides load into an endocytic vesicle, with peptide fragments that fuse vesicles containing MHC Class II molecules on the cell surface.

Synthesis and Assembly of MHC Class II

• The purpose of MHC class II molecules is facilitating the binding of peptides generated in intracellular vesicles in APCs to present these peptides to CD4 T cells.
• Proteins that create Class II, alpha and beta subunits, are translocated into the ER during synthesis.
• The ER is full of cytosolic peptides that are transported by TAP.
• The Invariant chain (li) binds to MHC class II to prevent peptide binding in the ER, and the section of the Invariant chain that binds the peptide-binding cleft is known as CLIP.
• Invariant chains also guide MHC class II molecules out of the ER into a vesicle that is part of the endocytic system.
• Vesicles are proteases that cleave Ii through acidification, and in the process the CLIP portion remains bound to the MHC molecule.
• A MHC-like molecule known as HLA-DM exists in the vesicle.
• It interacts with MHC Class II, causes a conformational change, and releases CLIP, which allows peptides to bind, and the complex is transported to the cell surface.

Termination of MHC Class II: Antigen Presentation

• Dendritic cells (DCs) carry out surveillance of antigens.
• Material is uptaken through micropinocytosis, normally loaded on MHC class II, and transported to the surface.
• The ubiquitin proteasome system continuously endocytoses/degrades uptaken peptides.
• March1, or E3 ubiquitin ligase, is responsible for targeting MHC Class II for degradation.
• DCs undergo maturation which shuts off the expression of the March1 gene.
• Maturation is obtained through signals coming from TLR-4.
• The antigen:MHC Class II complexes gather on the DCs' surface.

Cross Presentation Explained

• Dendritic cells capture antigens from infected or dying cells, such as virus-infected cells or tumor cells.
• Capture may be through phagocytosis, receptor-mediated endocytosis, or cellular debris uptake.
• Captured antigens are processed and degraded into smaller peptide fragments via enzymes in specialized compartments called endosomes and lysosomes.
• Typically peptides generated in the vesicular system load onto MHC class II molecules via cross-presentation.

Remember

• If cells display a foreign antigen on an MHC molecule, they also express B7 which binds to the Co-stimulatory receptor CD28 on T cells.
• B7 is always expressed after Pathogen Recognition Receptor activation.

Lymphocyte Receptor Signaling – T Cells Info

• Lymphocytes circulate through the blood, lymph, and lymphoid tissues looking for a specific antigen.
• It is converted to effector lymphocytes through stimulation/downstream pathways.

Principles of Signal Transduction

• A receptor links the extracellular environment with the interiors of a cell. Sense extracellular events and converts them into intracellular biochemical events.
• A ligand/ first messenger is introduced via an antigen to begin intercellular signaling cascades.
• Transduction occurs when there is a recruitment of of intercellular proteins to the membrane.
• Enzymatic proteins are activated to amplify the signal.
• Effector proteins modulate cellular responses to the ligand. Some examples of effectors are transcription factors and metabolic enzymes.

Transducers & second messengers

• Protein Kinases are the most common enzyme involved in signal transduction that catalyze phosphorylation.
• Phosphorylation = covalent attachment of a phosphate group from ATP to a protein.
• Dephosphorylation = detachable of a phosphate group that is carried out by phosphatases.
• The 2 types of kinases are serine/threonine kinases and tyrosine kinases.
• Adaptor proteins lack enzyme activity.
• Recruit and mediate assembly of signaling complexes at the cell membrane upon receptor activation via Activation which localizes proteins to the membrane.
• Assembly of signaling complexes involves interactions through protein-interaction domains such as SH2, SH3, and PH.
• SH2 binds phosphor-tyrosine.
• SH3 binds proline.
• PH binds phosphoinositides – a phospholipid.
• Small G proteins are also involved in signal transduction and they are membrane-bound.
• These small G proteins exist in 2 states.
  • Inactive = bound to GDP
  • Active = bound to GTP
• The exchange of GDP for GTP is mediated Guanine nucleotide Exchange Factors (GEFs).
• Intrinsic GTPase activity of small G proteins convert GTP into GDP causing them to become inactive.
• The process leading to the inactivation of small G proteins is accelerated by GTPase-activating Proteins (GAP).
• Signaling molecules are recruited in several mechanism:
  • Phosphorylation of the receptor or adapter proteins and recruits SH2-containing proteins
  • Activation of small G proteins and binds signaling molecules.
  • Modified membrane lipids Phosphokinases phosphorylate Phosphatidyl-inositol which then recruits PH domain-containing proteins.

Signal Amplification Details

• Many types:
  • Kinase Cascade: series of kinases that phosphorylate and activate each other.
  • Second Messengers are small molecules that are activated by enzymes to activate multiple target proteins.
• Signal strength indicates magnitude of the cellular response.
• Can be All-or-nothing or increase as the strength of the signal increases.

Signal Termination Info

• Several mechanisms occur:
  • Dephosphorylation by phosphatases
  • Polyubiquitination and degradation by the proteasome.
  • Common for membrane proteins is Monoubiquitination and degradation at lysosomes
• Dephosphorylation is reversible way to keep signals inhibited.
• Degradation is a long-lasting process.

T Cell Receptor Signaling

• The TCR is able to recognize but unable to signal, so it associates with the CD3 complex, which is needed for both signaling and proper expression of the TCR.

CD3 Complex Info

• CD3 Complex consists of CD3ε, γ, δ and ζ chains.
• Chain that do not contain extracellular Ig-like domains are the exception.
• All of them have intracellular Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), containing 2 tyrosine residues that get phosphorylated and begin signaling from the TCR.

TCR Sensitivity

• It can be activated through interaction with only 1 antigen:MHC complex.
• The 2 factors are: conformational changes and TCR clustering

Signaling Initiation

1. TCR and CD4/CD8 coreceptors interact with antigen:MHC complex.
2. The cytoplasmic domain of CD4/CD8 is associated with Tyrosine kinase, Lck.
3. Lck phosphorylates tyrosine on ITAMs of CD3 proteins and ζ chains.
4. ZAP70 is recruited and binds to phospho-tyrosines on ITA that occurs via SH2 domains.
5. Lck phosphorylates and activates ZAP70.
6. ZAP70 phosphorylates the adaptor proteins LAT and SLP-76.
7. Phospho-SLP-76 binds to LAT via Gads.
8. PI 3-kinase is recruited and makes PIP3.
9. The results in 4 downstream signaling pathways to profound changes in T cells.

Activation of Transcription Factors Info

• Activation of Transcription Factors
• Phospholipase C-y (PLC), an enzyme that divides phospholipids, is recruited to the membrane.
• The enzyme binds PIP3 through the PH domain and to phosphor-tyrosine in LAT through the SH2 domain.
• PLC is activated via the kinase Itk.
• When activated, PLC cleaves phosphatidyl inositol bi-phosphate (PIP2) into: Diacylglycerol (DAG) and inositol tri-phosphate (IP3).
• While DAG remains bound to the membrane, IP3 diffuses into the cytosol, binds to an IP3 receptor which causes Ca2+ to be released in the cytosol.
• Ca2+ levels lead to oligomerization of the ER protein, STIM1.
• STIM1 binds to the plasma membrane and directly interacts with CRAC (ORAI1).
• This opens the Ca2+ channel and causes Ca2+ to flow in.
• Transcription Factor, Nuclear Factor of Activated T cells or NFAT, promotes expression of genes.
• In naïve T cells, NFAT in the cytosol is phosphorylated until Ca2+ binds to Calmodulin and causes a conformational change that binds Calcineurin (a phosphatase).
• Finally, Calcineurin dephosphorylates the NFAT, freeing it.

Activation of the Transcription Factor

• 1. AP-1 is a heterodimer consisting of 2 proteins: c-Jun & c-Fos is is actived as follows.
• DAG recruits RasGRP. RasGRP is a GEF that activates the small G protein Ras through the exchange of GDP for GTP.
• The Sos, a GEF, recruites the membrane to LAT and activates Ras.
• 3-kinases relay when Ras is greatly activated and becomes "super activated".
• Mek activates Raf. RAF is a MAPK kinase kinase Kinase whereas Mek is a MAPK kinase Kinase.
• MAP kinase (Erk), or Erk, is activated via Mek, a kinase, that phosphorylates Elk which is a transcription factor.
• Expression of c-June and c-Fos is promoted as Phospho-Elk is moved inside the nucleus.
• As a result, a heterodimer known as AP-1 forms and promotes expression of T cell activation genes.

Activation of Transcription Factor, NF-KB

1. NFKB is sequestered in the cytosol bound to IKB
2. Dag recruits and activates Protein Kinase C (PKC)
3. Many proteins become phosphorylated.
4. One of these proteins phosphorylates IKB, which targets proteasomal degradation for the activation.

Integrin Adhesion

• Immune synapse structures are created, which stabilizies receptors and complexes, creating a region in the T cell that is direct contact with the APC or target cell.
• Integrin (LFA-1 heterodimer) binds integrin adhesion receptor (ICAM) and stabilizes signaling, but normally has dispersion and low affinity.
• ADAP, binds to LAT, and the rest of the complex is recruited to the membrane. = Small G Protein Rap1, activates the membrane, and induces a conformational change, increasing aggregation

Enhancing Cytoskeletal Reorganization

• This is needed for TCR and APC stabilization.
• Vav, or GEF, begins a membrane recruitment by working with PIP3, NKc, and SLP-76.
• GEF goes on to activate Cdc42 causing an additional conformational change.
• Activated Wasp then recruits WIP and Arp2/3. Polymerization takes place here, and the cytoskeleton reorganized.

Immune Synapse Details

• A Region of the T cell that is in with the APC or target cell.
• All receptors aggregate, or combine, into a central supramolecular activation complex.
• Peripheral surrounds the cSMAC

B Cell Receptor Signaling

• Like TCRs, BCRs cannot initiate receptor signaling on its own.

B Cell Receptor

1. BCR associates with a heterodimer complex (Iga and Igß).
2. 3 membrane bound kinases (Blk, Fyn and Lyn) associate with the receptor and phosphorylate the ITAM.
3. Multiple BCRs trigger signaling via multivalent antigens. These multivalent antigens aggregate on multiple BCRs, clustering and aggregating signaling to activate.
4. As a tyrosine, Syk goes and binds to ITAM as a tandem.
5. Syk continues to phosphorylate several proteins in the signal. These proteins are recruited to the SLP-65, promoting further signal transmission.

B Cell Co-receptors

• B cell coreceptors can enhance the signaling.
• B Cell Co-receptor: Consists of CD19, CD21 and CD81, enhance B-cell signaling.
  • CD21 binds the break-down of C3b (product of C3b cleavage), remains bound
  • CD21 - Complement receptor 2
  • CD81= not known function
  • CD19= get phosphorylated (Blk, Fyn or Lyn and recruits PI-3 kinase)

PI-3-Kinase Notes

• PI-3 kinase, generates PIP3 which encourages cell survival and is used to activate transcription factors and cytoskeletal reorganization.

Modulation of Antigen Recognition

• Full naive lymphocyte activation requires additional signals.
• The Receptors in the membrane of b and T and they provide secondary signals, for additional antigen receptors. (ex CD28 in T, and CD40 B.)
• Additional enhancement to antigen signals.

B and T Signlaing Regulation

• In contrast, the suface of some be and T cells are other receptors and they down regulatesignaling to prevents excessive immune repsosne.
• These are down regulating.
• Downregulation prevents excess activation.

CD28 receptors facts

• Expressed naive T and can be b7 in APC, has Costulatory signals
• Expressed naive T and can be b7 in APC, has Costulatory signals
• B7 (CD80/CD86) is pathogen expression in APC
• For total Activation, TCR and CD28 must be together
• PI3K phosphorylates the CD28 receptor, activating 3 down sream signaling pathway
• CD 28 produces optimal transcription with full IL2 expression. Full 2 is needed for cell and life survival.

B Cell Co-activation points

• For the production of B cells, coactivation, can come directly from pahtogenic PRRs or from 4 + T cells
• Provides for co-activtion using lymphokines and lymphokine for interaction.

Contolling B Cells

• CTLA 4 can control active T through production of interferons
• A A with high affect can bind c7 with a high interaction
• Can recruit phosphatases.

Immuno Receptor Notes

• These receptors have intracellular Immunoreceptor Tyrosine-based Inhibition Motifs

All Lymphocytes Origins

• Are made are of multipotent stem
• B cells originate for maro and t cells start in thymo
• The develoope t into phases each with different factors exresion factors
• The eariler starts in B Cells
• Here the cell and adhesion mols
• Contact the hematopoietic stem cells

Cell Development

• PU.1: low levels
• Ikaros, Differentiation occurs for cells for CLP( common lymphoid pogentitor cell)
• Stromal interactions happen, stromal receports promote IL7 receptors,
• IL7 receptor is needed for factor production through the express of Pax (B cell growth gen)
• Promotes expression for CO-receptors for CB19